Antigen

Antigen Class

TFs and others

Antigen

Xbp1

Cell type

Cell type Class

Liver

Cell type

Liver

MeSH Description

A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.

Sample

source_name

liver: 48h after sham operation

strain

C57Bl6-J

tissue

liver

genotype

wildtype

chip antibody

anti-XBP1 M-186 (sc-7160-X, Santa Cruz, Heidelberg, Germany)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Liver tissue was washed in PBS, cut into pieces ≤ 2mm diameter and fixed for 15 min in 1% of formaldehyde (Sigma). Tissue was washed three times in PBS and stored at -80°C. Upon thawing, tissue was incubated in 125 mM glycine (Sigma) for 5 min at room temperature, washed in PBS and nuclei were prepared on ice using a dounce homogenizer. All following steps were performed on ice, if not specified otherwise. Nuclei were washed in PBS, resuspended in ChIP buffer (4 ml of buffer per 0.5 ml of tissue; 100mM NaCl, 133mM Tris pH 8.0, 5 mM EDTA, 0.2% NaN 3 ,0.67% SDS, 1.67% Triton X-100) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche, Mannheim, Germany) and phosphatase inhibitors (50 mM NaF,1 mM β-GP, 1 mM NaV). Chromatin was sonicated to an average length of 300 bp, pre-cleared once with protein A-Sepharose and once with protein G-sepharose beads, both blocked with 0.5% E. coli tRNA (Sigma) and 0.5% BSA. For immunoprecipitating XBP1 protein, we used anti-XBP1 M-186 (sc-7160-X, Santa Cruz, Heidelberg, Germany). 10 µg of antibody were incubated overnight with 2ml of chromatin. Pre-blocked proteinG-sepharose beads were added to the antibody/chromatin mix, incubated for 2-3h and washed as follows: 3 times with mixed micelle buffer (150 mM NaCl, 20 mM Tris pH 8.1, 5 mM EDTA, pH 8.0,5.2% sucrose, 0.02% NaN3 , 1% Triton X-100, 0.2% SDS), 2 times with buffer-500 (50 mM HEPES pH 7.5, 0.1% sodium deoxycholate, 1 mM EDTA, 500 mM NaCl, 1% TritonX-100, 0.2% NaN 3 ), 2 times with LiCl/detergent buffer (10 mM Tris pH 8.0, 0.5% sodiumdeoxycholate, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.2% NaN3 ) and once with TE. DNA was reverse-crosslinked overnight (2% SDS in TE, 65°C), purified using QiaQuickcolumns (Qiagen, Madrid, Spain) and quantified using PicoGreen (Fisher Scientific, Madrid, Spain). 2-5 ng of ChIP DNA were used for ChIPseq library preparation as described in the reference given below. Blecher-Gonen R1, Barnett-Itzhaki Z, Jaitin D, Amann-Zalcenstein D, Lara-Astiaso D, Amit I., High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states. Nat Protoc. 2013 Mar;8(3):539-54

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

28833104

Reads aligned (%)

85.3

Duplicates removed (%)

21.3

Number of peaks

3197 (qval < 1E-05)

mm9

Number of total reads

28833104

Reads aligned (%)

85.0

Duplicates removed (%)

21.3

Number of peaks

3207 (qval < 1E-05)